CN1735684A - 用于犬和人埃利希病的免疫诊断的p153和p156抗原及其用途 - Google Patents
用于犬和人埃利希病的免疫诊断的p153和p156抗原及其用途 Download PDFInfo
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Abstract
从犬埃利希氏体(p153基因)和恰菲埃利希氏体(p156基因)克隆了编码两种免疫反应性糖蛋白的序列。这两种糖蛋白是种特异性的免疫反应性直向同源物，可以用作犬埃利希氏体和恰菲埃利希氏体的亚单位疫苗和用于血清学和分子诊断。
Description
相关申请的交叉引用关系
本申请要求美国临时专利申请60/423573的权益，该申请提交于2001年11月4日，现已放弃。
联邦资助说明
本发明部分依靠联邦政府提供的资金，即国立过敏和传染病研究所(National Institute of Allergy and Infectious Diseases)基金编号：AI31431而完成。因此，联邦政府对于本发明享有一定权利。
发明背景
技术领域
本发明主要涉及分子和免疫诊断领域。更具体地，本发明涉及来源于犬埃利希氏体(Ehrlichia canis)和恰菲埃利希氏体(Ehrlichia chaffeensis)的种特异性免疫反应蛋白的直向同源物(～200kDA)，用于种特异性诊断犬埃利希病和人嗜单核细胞埃利希病。
相关技术的描述
犬单核细胞埃利希病是一种潜在的致死性蜱传播狗类疾病，该疾病主要是由立克次氏体(犬埃利希氏体(Huxsoll等人，1970))引起，在世界范围内广泛分布。犬埃利希氏体是一种专性细胞内细菌，表现出对单核细胞和巨嗜细胞的嗜性(Nyindo等人，1971)，能在脊椎动物宿主体内引起持续感染(Harrus等人，1998)。该病的特征分三个阶段：持续2-4周的急性感染阶段；亚临床感染阶段，在此阶段狗可能维持感染好几年，但不显现出任何临床症状；此后是慢性阶段，在此阶段由于骨髓再生不良，很多狗的病况进行性恶化预后更差(Troy等人，1990)。
犬埃利希氏体感染犬科动物引起埃利希病。犬埃利希病包括急性阶段和慢性阶段。急性阶段的特征为发热、浆状的眼鼻排泄物、厌食、抑郁、和体重减轻。慢性阶段的特征除了更严重的急性病临床症状外，还有严重的全血细胞减少症、鼻出血、血尿、粪便带血。如果在疾病进程早期进行治疗，狗对于强力霉素的治疗反应良好。然而，慢性感染的狗对于该抗生素反应不佳。因此，早期诊断对于犬埃利希病的治疗尤为重要。
急性阶段治疗此疾病对其最佳预后很重要。血液学异常(诸如：白血球减少症和血小板减少症)常常可为犬埃利希病提供有用的证据，也是最初诊断的重要因素(Troy等人，1990)。然而，因为犬埃利希病的临床表现是非种特异性的，这给诊断带来了困难。
用血清学方法，如：间接荧光抗体(IFA)检测，来诊断犬埃利希病由于其简便、可靠和成本效应(cost effectiveness)低已经成为标准的方法(Troy等人，1990)。然而，间接荧光抗体检测的缺点包括：该方法不能做出种特异性诊断，因为它与也感染狗的其它密切相关的埃利希体属类(恰菲埃利希氏体，E.ewingii，Anaplasma phagocytophilum，以及A.platys)会发生抗原交叉反应。主观的解释也可能由于交叉反应性抗原而得到假阴性或假阳性结果。据报道，已开发的种特异性检测犬埃利希氏体的其它诊断方法，如聚合酶链式反应(PCR)，比细胞培养分离法更灵敏，但是这种方法需要专门训练和昂贵的仪器(McBride等人，1996)。病原体的分离很费时，仅有少数几家实验室一直成功地使用这一方法。而且，采用这一方法来确定具体的病因，还需要其它检测方法来鉴定该分离物。
已报道犬埃利希氏体和恰菲埃利希氏体之间存在血清学交叉反应性抗原。一些主要的血清学交叉反应性蛋白的分子量为28-30kDa(Chen等人，1997；Rikihisa等人，1994)，现在已知这些蛋白是由同源性多基因家族所编码(Ohashi等人，1998a，b)。在犬埃利希氏体和恰菲埃利希氏体中分别有22个和25个基因同源但不完全相同，而p28基因相同并进行了测序。在犬埃利希氏体和恰菲埃利希氏体的P28蛋白之间观察到类似的种内和种间菌株的同源性，从而解释了这些蛋白所产生的血清学交叉反应性(McBride等人，1999)。
最近一项报道表明恰菲埃利希氏体的rP28蛋白在诊断人类嗜单核细胞埃利希病(HME)中不敏感(Yu等人，1999a)。其根本原因似乎是不同株的恰菲埃利希氏体中P28蛋白有变异性(Yu等人，1999b)。与之相反，分布于不同地域的犬埃利希氏体中鉴定到的P28基因却是保守的，而且证明犬埃利希氏体的rP28可用于犬埃利希病的诊断(McBride等人，1999；Ohashi 1998a)。已克隆了其它同源免疫反应性蛋白(包括犬埃利希氏体(gp140)和恰菲埃利希氏体的糖蛋白(gp120))(Yu等人，1997，2000)。恰菲埃利希氏体的rgp120的反应性与用于嗜人单核细胞埃利希病血清诊断的间接荧光抗体具有很好的相关性，而且采用犬埃利希氏体的rgp140的初步研究提示它可能是一种敏感可靠的免疫诊断抗原(Yu等人，1999a，2000)。
以往技术缺乏用于犬埃利希氏体和恰菲埃利希氏体的血清学和分子诊断的特异性抗原，以及缺少采用这些诊断抗原的方法。本发明填补了该领域长久已来的需求。
发明概述
已鉴定了犬埃利希氏体的一种强免疫反应性的43kD蛋白(p43)(美国专利号：6355777)。作为一种免疫诊断抗原，p43蛋白与间接荧光抗体检测相比具有96％的准确性，为犬埃利希氏体感染提供了种特异性的诊断方法。进一步的研究显示犬埃利希氏体的p43代表了一种推测分子量为153kD蛋白质的N末端部分，是报道的埃利希氏体属类中的最大的免疫反应性蛋白质。用蛋白凝胶电泳分析重组表达的p153蛋白片段，结果显示其分子量大于预测的分子量(～10至30％)，在N-和C-末端片段中存在聚糖(carbohydrate glycan)，表明p153是一种糖蛋白。
对可获得的恰菲埃利希氏体基因组序列(95％)进行了BLASTn搜索，在恰菲埃利希氏体中鉴定到编码p153蛋白直向同源物(orthologh)的基因。犬埃利希氏体的p153基因(4263bp)和恰菲埃利希氏体的p156基因(4389bp)具有相似的染色体定位，在同源性(～87％)脱氧鸟苷三磷酸三磷酸水解酶基因以及开放阅读框之前同源性(～90％)的基因间序列的下游。在该糖蛋白基因之间观察到了核酸序列的同源性(50％)，这支持了先前有关p43基因片段遗传多样性的发现，而p153和p156蛋白的氨基酸序列相似性为32％。分子量为200kD的天然犬埃利希氏体蛋白能与用p153N-末端区域(p43)所产生的抗血清反应，提示该天然蛋白已经过翻译后修饰。类似地，包含恰菲埃利希氏体p156蛋白N-末端区的重组蛋白比预计的(～200kD)迁移更多，而且在该重组蛋白上还测到了碳水化合物。在这段N-末端片段上鉴定到有一个主要的免疫反应表位。染色体定位、氨基酸的同源性、以及生物物理特征都支持以下结论：即p153和p156糖蛋白(命名为gp200s)是种同源性免疫反应性直向同源物。
在犬埃利希氏体p153蛋白的N-(P43)和C-末端区域以及恰菲埃利希氏体p156直向同源物的N-末端区域中所鉴定出的主要免疫反应性表位，可用于血清学诊断和疫苗。而且，编码这些蛋白的基因是种特异性的，可用于分子诊断学的发展。
本发明的其它和更深入的方面、特征、和优点，将通过以下本发明提供的优选实施例描述得以显现。给出这些实施例的目的在于说明。
附图简要描述
可以获得并详细了解上述的本发明特征、优点和目的、以及其它一些显而易见的特征、优点和目的。参考附图中加以解释的某些实施例，可以对上面简要概括的本发明进行更具体的阐述。这些附图也是本说明书的一部分。然而，须注意，附图用于说明本发明的优选实施例，因而不认为是对于发明范围的限制。
图1A和1B显示恰菲埃利希氏体p156(最上面一行)和犬埃利希氏体p153(最下面一行)蛋白质直向同源物的Lipman-Pearson氨基酸序列排列对比。氨基酸的相同性，保守替换(：)和半保守替换(.)表示在中间。
图2A和2B显示犬埃利希氏体p153(A)和恰菲埃利希氏体(B)重组蛋白片段用抗V5抗体检测得到的表达情况。犬埃利希氏体p153，泳道1；N-末端片段(1107bp，nt-1-1107)，泳道2，内部片段(910bp，nt1080-1990)；泳道3，内部片段(1000bp，nt-1950-2950)；和泳道4，C-末端片段(1280bp，nt-2940-4220)。恰菲埃利希氏体p156，泳道1，N-末端片段(1545bp，nt-125-1675)；泳道2，内部片段(1365bp，nt-1685-3050)；和泳道3，C-末端(1365bp，nt-2950-4315)。
图3A显示犬埃利希氏体10p153重组片段的Western免疫印迹。泳道1，N-末端片段(1107bp，nt-1-1107)；泳道2，内部片段(910bp，nt1080-1990)；泳道3，内部片段(1000bp，nt-1950-2950)，和泳道4，C-末端片段(1280bp，nt-2940-4220)。
图3B显示在对应于犬埃利希氏体p153的纯化重组片段上检测到碳水化合物，该重组片段用pRSET表达载体在大肠杆菌内表达。将结合于该重组蛋白的聚糖进行氧化并用生物素标记后，用链霉亲和素-碱性磷酸酶检测。
图4A显示恰菲埃利希氏体的p156重组片段(泳道1-3)与人血清(左图)和狗血清(右图)的Western印迹图。泳道1，恰菲埃利希氏体p156的N-末端片段(1545bp，nt-125-1675)；泳道2，内部片段(1365bp，nt-1685-3050)；以及泳道3，C-末端(1.365bph nt-2950-4315)。所表达的重组蛋白代表约95％恰菲埃利希氏体p156蛋白。
图4B显示三种对应的重组恰菲埃利希氏体p156蛋白(泳道1-3)的碳水化合物的检测。
图5显示犬埃利希氏体全细胞裂解物中蛋白质与来自犬埃利希氏体感染狗的多克隆抗血清的Western印迹图(泳道1)，和与抗重组p43(gp200)多克隆兔血清的Western印迹图(泳道2)，及与抗重组gp140多克隆兔血清的Western印迹图(泳道3)。
本发明的具体描述
据先前报道，犬埃利希氏体p43基因序列是1173bp(美国专利：6355777)，但是进一步分析揭示DNA测序错误导致产生一个人造中止密码子，和截短的基因序列。采用引物-接头基因步移方法，在原有p43克隆中2.4kbp片段的下游又确定了另外一个4.5kbp序列。不完整的p43基因序列补全后，显示了一个4263bp的开放阅读框，其编码预计分子量为153kD的蛋白质(命名为p153)。p153基因的上游有一个编码脱氧鸟苷三磷酸三磷酸水解酶的开放阅读框，p153基因前还有一段基因间非编码区，它们在犬埃利希氏体和恰菲埃利希氏体之间具有很高的同源性(分别为87％和90％)。
对恰菲埃利希氏体基因组和2.4kbp的p43克隆进行BLASTn搜索鉴定到高度同源性的核苷酸序列。关于该上游同源性编码序列和基因间核苷酸序列，在相同的染色体位置上发现了与犬埃利希氏体p153大小几乎相等的一个大开放阅读框(4389bp)，该阅读框编码的蛋白质具有预计的分子量156kD(p156)。发现犬埃利希氏体p153和恰菲埃利希氏体p156基因之间具有核酸序列同源性(约50％)；然而，该蛋白质的氨基酸全序列的相似性仅为32％(图1)。
在大肠杆菌中表达的基因构建物，代表了恰菲埃利希氏体p156蛋白(nt-125-1670；nt-1685-3050；nt 2950-4315)和四个犬埃利希氏体p153蛋白的片段(nt-1-1107(p43)；nt-1080-1990；nt-1950-2950；nt-2940-4220)，这些构建物在大肠杆菌中进行表达。犬埃利希氏体N-末端(nt 1-1107)和C-末端(nt-2940-4220)重组表达蛋白表现出很强的免疫反应性(图3A)。然而，恰菲埃利希氏体p156只有N-末端片段(nt-125-1670)具有免疫反应性(图4A)。
犬埃利希氏体(nt-1-1107和nt-2940-4420)和恰菲埃利希氏体p156重组蛋白片段(nt-125-1607)在SDS-PAGE上的迁移率大于预计值，表明该片段发生过翻译后修饰。随后，在犬埃利希氏体p153和恰菲埃利希氏体p156肽片段上检测到碳水化合物(图3B和4B)。
抗-p43抗体能与犬埃利希氏体全细胞裂解物中的一个约200kD的天然蛋白反应。而且，该200kD的蛋白也可被犬埃利希氏体感染的狗血清所识别(图5)。先前鉴定为p43(p153的N-末端部分)的一部分基因序列在GenBank中被指定为登录号AF252298。修正后编码p153的序列在GenBank中被指定为登录号AY156950。
染色体定位、氨基酸同源性、和生物物理性质支持以下结论，即p153和p156糖蛋白(命名为gp200s)是种特异性的免疫反应性直向同源物。这些蛋白对疫苗的开发具有潜在的用途，也可用作灵敏可靠的血清诊断用抗原来诊断埃利希氏体属感染。先前的发现也支持这一点，该发现表明犬埃利希氏体p43具有免疫反应性，用作血清诊断性抗原(美国专利：6355777)。与抗p43抗体之间的反应和间接荧光抗体滴度(IFA)＞40的样品之间，具有100％的相关性，并能和间接荧光抗体滴度＜40的数个样品发生反应。几份间接荧光抗体阴性的样品能与p43抗体产生弱反应，这提示p43蛋白可能是一种更为敏感的血清诊断性抗原。本发明所提供的结果表明p43是更大的犬埃利希氏体p153蛋白的一部分。
本发明涉及分离的编码犬埃利希氏体免疫反应性表面蛋白p153和恰菲埃利希氏体p156蛋白的多聚核苷酸。优选该分离的多聚核苷酸编码具有SEQ IDNO：1和2中所示氨基酸序列的蛋白质。此外，由于遗传密码的简并性，该DNA可以在核苷酸序列上有所不同。
本发明还包括含有这些分离的多聚核苷酸和在细胞中表达该DNA所必须的调节序列的载体；分离和纯化的p153和p156蛋白质；以及抗这些蛋白质的抗体。
本发明还涉及p153和p156蛋白在制备犬和人埃利希病疫苗中的用途。此外，还提供通过检测狗血清是否与该p153或p156蛋白反应来确定狗或人是否感染了埃利希氏体属类的方法。所用的蛋白可以是重组的，可用Western印迹分析来检测血清对这些蛋白质的反应。由于和先前分离的犬埃利希氏体p28蛋白的反应也是犬埃利希氏体感染的可靠标志，诊断可以包括检测对犬埃利希氏体p153蛋白、gp140蛋白、和p28抗原的免疫反应性。
本发明还涉及测定狗或人是否被埃利希氏体属类感染的血清学诊断试剂盒。该试剂盒包括本文所述的固定化蛋白(p153或p156)、合适的狗血清稀释缓冲液、连接有报告分子的抗狗血清第二抗体、和检测该报告分子的合适的试剂。可行的固定抗原的方法包括将抗原连到膜或者微量滴定板上。报告分子可以是荧光素酶、辣根过氧化酶、β-半乳糖苷酶、或荧光标记。
本发明还涉及测定狗是否被埃利希氏体属类感染的PCR扩增方法。提取潜在感染的狗或人的血液DNA，用犬埃利希氏体p153基因或恰菲埃利希氏体p156基因的特异性寡核苷酸引物进行PCR扩增。所得的PCR扩增产物用凝胶电泳等方法按大小分离，检测到适当大小的产物表明已感染了埃利希氏体属类。
本发明还涉及一套PCR检测p153和p156基因的试剂盒。该试剂盒包括提取血液DNA的试剂、p153或p156的特异性寡核苷酸、和用于PCR扩增的试剂。
依照本发明，可以采用本领域技术人员所用的常规分子生物学、微生物学、和重组DNA技术。文献中对这些技术的详细说明。参见，例如，Maniatis，Fritsch&Sambrook，“分子克隆：实验手册(1982)”；“DNA克隆：实用方法，”卷I和II(D.N.Glover编著1985)；“寡核苷酸合成”(M.J.Gait编著1984)；“核酸杂交”(B.D.Hames & S.J.Higgins等编著(1985))；“转录和翻译”(B.D.Hames & S.J.Higgins等编著(1984))；“动物细胞培养”(R.I.Freshney，编著(1986))；“固定化细胞和酶”(IRL PRESS，(1986))；“分子克隆实用指南”(1984)。
本文所用的术语“宿主”不仅包括原核细胞，还包括酵母、植物和动物细胞等真核细胞。可用编码本发明蛋白的重组DNA分子或基因以任何本领域普通技术人员熟知的任何技术转化宿主。原核宿主可以包括大肠杆菌、鼠沙门氏菌、粘质沙雷氏菌、和枯草芽孢杆菌。真核宿主包括酵母菌(如：毕赤巴斯德酵母菌)、哺乳动物细胞和昆虫细胞。
通常，把含有能促进插入的DNA片段的有效转录的启动子序列的表达载体和宿主结合使用。该表达载体通常含有复制启始区、启动子、中止子、以及能够提供转化细胞表型筛选的特定基因。可用本领域已知的方法发酵培养转化的宿主以达到最佳细胞生长。可以用本领域技术人员熟知的方法来构建含有合适的转录和翻译调控信号的表达载体。例如，参见Sambrook等人，1989，分子克隆：实验手册(第二版)(冷泉港出版社，纽约)中所述的技术。
本文所用的术语“引物”指一种寡核苷酸，可以是天然存在的(如存在于纯化的限制性消化片段中)或是人工合成的，该寡核苷酸能在合适的条件下(即可诱导引物延伸而合成与核酸链互补的片段)作为合成起始点。所述条件包括：存在核苷酸和诱导试剂(如：DNA聚合酶)以及适当的温度和pH。引物可以是单链或双链的，必须足够长而能在诱导试剂存在下引发所需的延伸产物的合成。引物的确切长度取决于很多因素，包括温度、引物来源和使用的方法。例如，应用于诊断时，根据靶序列的复杂性，寡核苷酸引物一般包含15-25个或更多个核苷酸。也可使用核苷酸数目较少的引物。
本文选用的引物与特定靶向DNA序列的不同链“基本”互补。这就意味着该引物必须足以与其各自的链互补杂交。因此，引物序列不需要反映模板的确切序列。例如，可将非互补性核苷酸片段结合于该引物的5’末端，而引物序列的其它部分可与该链互补。或者，可将非互补碱基或更长的序列插入该引物中，只要该引物序列与所述序列足够互补或能与其杂交，形成合成延伸产物的模板。
给出以下实施例目的是说明本发明的各种实施方案，而不意味着以任何方式限制本发明。
实施例1
犬埃利希氏体p153和恰菲埃利希氏体p156蛋白质的特征鉴定
如前所述(McBride等人，2001；美国专利号：6355777)，从Lambda Zap II表达库中鉴定到的犬埃利希氏体p43蛋白基因。最初的2.4-kb克隆包含一个编码脱氧鸟苷酸-三磷酸三磷酸水解酶基因的开放阅读框(ORF)和下游截短的、位于p43基因片段之前的229bp的基因间序列。采用引物-接头PCR法以犬埃利希氏体的基因组DNA(Jake，北卡罗莱那株)作为模板，测定该p43开放阅读框的全长序列。扩增子直接以用于扩增的引物测序或克隆到TOPO/TA中进行序列分析。用犬埃利希氏体(E.canis)p43的全长克隆(2.4-kb)进行恰菲埃利希氏体基因组序列的BLASTn检索鉴定，得到恰菲埃利希氏体的直向同源物(p156基因)。
将犬埃利希氏体p153和恰菲埃利希氏体p156基因分成长片段(1至1.5kbp)，克隆到pUni/V5-His-TOPO Echo供者载体中，并与pBAD Thio-E或pRSETEcho受者表达载体重组。用阿拉伯糖或IPTG诱导后，重组蛋白表达4小时。膜标记后，用检测糖蛋白的免疫印迹试剂盒(Bio-Rad)对所表达的重组蛋白进行聚糖检测。在大肠杆菌中表达前面所述的恰菲埃利希氏体重组Dsb蛋白(McBride等人，2002)，作为对埃利希氏体进行糖蛋白检测研究时的阴性对照蛋白。将犬埃利希氏体全细胞裂解物用梯度胶(4-12％Bis-Tris，Novagen)进行凝胶电泳分离，用半干转移单元(Bio-Rad)(semidry transfer unit)转移至干净的硝酸纤维膜上。如前所述(McBride等人，2001)进行免疫印迹分析。
讨论
含有犬埃利希氏体p153N-末端(p43)部分的克隆具有很强的免疫反应性，使得该克隆得到最初鉴定并分析(McBride等人，2001)。与间接荧光抗体试验检测狗犬埃利希氏体抗体的结果相比，p43表现出优良的敏感性和特异性。而且，由于抗重组p43多克隆抗体并不与恰菲埃利希氏体感染的DHS2细胞反应，因此p43似乎能够提供种特异性检测。在恰菲埃利希氏体中鉴定到p153的直向同源物(p156)，此直向同源物(也称为定向进化同源物)是基因多样性的表现，氨基酸同源性程度低，支持了先前的发现(即p43蛋白是种特异性抗原)，p43也因此可能是极好的种特异性免疫诊断性抗原。主要的线性B细胞抗原表位存在于p153蛋白的N-(p43)区域和C-末端区域。
P43重组蛋白所显示的分子量大于预期值(约30％或约10kD)，这一点是最初并未认识到的。先前报道的埃利希氏体的糖蛋白gp120和gp140比预期值大60％至100％。尽管(电泳时)分子量的漂移程度要小得多，但p43蛋白是一种糖蛋白，这在碳水化合物检测中得到了附着聚糖的证实。与p43蛋白的发现相一致的是，表达的恰菲埃利希氏体p156重组基因片段表现的分子量大于预期值，而且在这些片段上检测到了碳水化合物。此外，犬埃利希氏体p153的C-末端片段也表现出分子量大于预期值(约10％或6kD)。
当鉴定出p43基因时，从全细胞裂解物中对应的犬埃利希氏体天然蛋白质却不与抗p43抗血清反应。根据本发明提供的发现，这一分歧可能归因于以下事实：即p43基因代表一个不完全的开放阅读框，而该阅读框并不编码43kD蛋白。此外，由于此蛋白分子量很大(＞150kD)，故需对凝胶电泳的条件给予特别注意才能获得免疫印迹法对该蛋白的鉴定结果相一致。犬埃利希氏体全细胞裂解物中的200kD蛋白与抗p43多克隆抗体起很强的免疫反应。该蛋白的分子量与预测的偶联了某些聚糖使其分子量有所增加的p153的分子量相一致。这一发现同样与恰菲埃利希氏体p156重组片段几乎代表整个开放阅读框的分子量相一致。
埃利希氏体属类的糖蛋白是在致病细菌中最早被特征鉴定的此类蛋白质。至今已发现的埃利希氏体的糖蛋白，可被受感染的病人和动物的抗体一致和强烈地识别。这些独特的暴露于表面的免疫反应性蛋白质在疫苗开发中具有潜在价值，而且这些蛋白可以是亚单位疫苗的重要组分。
本文中引用了以下参考资料：
Chen，等人，1997.采用Western免疫印迹法分析罹患嗜人单核细胞埃利希病的病人的抗体应答，该病可以由不同株的恰菲埃利希氏体和犬埃利希氏体(Ehrlichia canis)所引起CIin.Diagn.Lab.Immunol.4：731-735.
Harrus，等人，1998.感染犬埃利希氏体后20-34个月的狗的埃利希氏体DNA的扩增.J.Clin.Microbiol.36：73-76.
Huxsoll，D.L.，P.K.Hildebrandt，和R.M.Nims.1970.热带犬全血细胞减少症.J.Am.Vet.Med.Assoc.157：1627-1632.
McBride，等人，1996.狗急性犬埃利希氏体感染的PCR检测.J.Vet.Diagn.Invest.8：441-447.
McBride，等人，1999.Clin.Diag.Lab.Immunol.6：392-399.
McBride，等人，2001.用重组蛋白对犬埃利希氏体感染的免疫诊断.J.Clin.Microbiol.39：315-322.
McBride，等人，2002.埃利希氏体属类的一种免疫反应性DsbA样硫-二氧化硫氧化还原酶的鉴定和功能分析。Infect.Immun.70：2700-2703.
Nyindo，等人，1971.热带犬全血细胞减少症：病原体--犬埃利希氏体的体外培养.Am.J.Vet.Res.32：1651-1658.
Ohashi，等人，1998a.编码犬埃利希氏体的免疫显性的30kD的主要外膜蛋白的多基因的克隆和特征分析以及该重组蛋白在血清学诊断中的应用。J.Clin.Microbiol.36：2671-2680.
Ohashi，等人，1998b.多态性多基因家族编码的恰菲埃利希氏体的免疫显性主要外膜蛋白.Infect.Immun.66：132-139.
Rikihisa等人，1994.恰菲埃利希氏体，犬埃利希氏体，或E.ewingii感染狗和人的Western免疫印迹分析.J.Clin.Microbiot.32：2107-2112.
Troy，G.C.and S.D.Forrester.1990.犬埃利希病，p.404-4 18.C.E.Green编著，狗和猫的传染性疾病.W.B.Sauders Co.，Philadelphia.
Yu，等人，1997.编码恰菲埃利希氏体的120kDa免疫显性蛋白的基因的克隆和测序.Gene 184：149-154.
Yu，等人，1999a.恰菲埃利希氏体重组蛋白用于嗜人单核细胞埃利希病血清学诊断的比较。J.Clin.Microbiot.37：2568-2575.
Yu，等人，1999b.从人体中分离的恰菲埃利希氏体28-kD外膜蛋白基因的遗传多样性.J.Clin.Microbiol.37：1137-1143.
Yu，等人，2000.编码犬埃利希氏体120-kD蛋白的基因的分子克隆和特征鉴定以及将该重组120-kD蛋白应用于犬埃利希病的血清学诊断.J.Clin.Microbiol.38：369-374.
在本说明书中提及的任何专利或出版物代表本发明涉及领域技术人员的水平。这些专利和出版物纳入本文作为参考，就好像各篇出版物被单独而具体地纳入作为参考一样。
本领域的技术人员不难理解：本发明十分适于实施其目标，并获得所述的和其本身所有的结果和优点。这些实施例连同本文所述的方法、步骤、处理、分子，和特殊的化合物以优选实施例为代表，都是示范性的，并不用于限制本发明范围。在不违背所附权利要求书范围所界定的本发明的精神下，本领域技术人员可以进行改动和作为其它用途。
序列表
<110>J.W.麦克布里奇(MCBRIDE，JERE W.)
D.H.沃克(WALKER，DAVID H.)
<120>用于犬和人埃利希病的免疫诊断的P153和P156抗原及其用途
<130>CLFR：235W0
<140>PCT/US2003/034916
<141>2003-11-04
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<170>PatentIn Ver.2.1
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Claims (26)
1.一种编码犬埃利希氏体免疫反应性表面蛋白p153的DNA，其特征在于，所述DNA选自下组：
(a)分离得到编码具有SEQ ID NO：2氨基酸序列的p153蛋白的DNA；和
(b)分离得到的编码所述蛋白的DNA，其中所述DNA的序列由于遗传密码子简并性而在10个密码子序列上不同于(a)中分离得到的DNA序列。
2.一种载体，其特征在于，含有权利要求1中所述的DNA和在细胞中表达该DNA所必须的调控元件。
3.如权利要求2所述的载体，其特征在于，所述的DNA编码的p153蛋白具有SEQ ID NO：2所示的氨基酸序列。
4.一种用权利要求2所述载体转染的宿主细胞，其特征在于，所述载体编码具有SEQ ID NO：2所示氨基酸序列的p153蛋白。
5.如权利要求4所述的宿主细胞，其特征在于，所述细胞选自下组：细菌细胞、哺乳动物细胞、植物细胞和昆虫细胞。
6.一种分离纯化的犬埃利希氏体的免疫反应性表面蛋白p153，其特征在于，该蛋白由选自下组的DNA所编码：
(a)分离得到的编码具有SEQ ID NO：2氨基酸序列的p153蛋白的DNA；和
(b)分离得到的DNA，其在密码子序列上由于遗传密码子简并性而不同于(a)中分离得到的DNA序列。
7.一种编码恰菲埃利希氏体免疫反应性表面蛋白p156的DNA，其特征在于所述DNA选自下组：
(a)分离得到的编码具有SEQ ID NO：1氨基酸序列的p156蛋白的DNA；和
(b)分离得到的编码所述蛋白的DNA，其中所述DNA的序列由于遗传密码子简并性而在密码子序列上不同于(a)中分离得到的DNA。
8.一种载体，其特征在于，含有权利要求7所述的DNA和在细胞中表达该DNA所必须的调控元件。
9.如权利要求8所述的载体，其特征在于，所述DNA编码的p156蛋白具有SEQ ID N0：1所示氨基酸序列。
10.一种用权利要求8所述载体转染的宿主细胞，其特征在于，该载体编码的p156蛋白具有SEQ ID NO：1所示氨基酸序列。
11.如权利要求10所述的宿主细胞，其中所述细胞选自下组：细菌细胞、哺乳动物细胞、植物细胞和昆虫细胞。
12.分离并纯化的恰菲埃利希氏体免疫反应性表面蛋白p156，其特征在于，该蛋白由选自下组的DNA所编码：
(a)分离得到的编码具有SEQ ID NO：1所示氨基酸序列的p156蛋白的DNA；和
(b)分离得到的DNA，其在密码子序列上由于遗传密码子的简并性而不同于(a)中分离得到的DNA。
13.一种抗体，其特征在于，该抗体针对权利要求6所述的p153蛋白。
14.一种抗体，其特征在于，该抗体针对权利要求12所述的p156蛋白。
15.一种犬埃利希病疫苗，其特征在于，该疫苗含有权利要求6所述的p153蛋白。
16.一种犬埃利希病疫苗，其特征在于，该疫苗含有权利要求12所述的p156蛋白。
17.一种测定狗是否感染了埃利希氏体的方法，其特征在于，该方法包括以下步骤：
测定所述狗血清是否与犬埃利希氏体p153蛋白或恰菲埃利希氏体p156蛋白反应，其中能与p153蛋白或p156蛋白反应则表示所述的狗分别感染了犬埃利希氏体或恰菲埃利希氏体。
18.如权利要求17所述的方法，其中所述的蛋白是一种重组蛋白。
19.如权利要求17所述的方法，其中采用Western印迹分析来测定所述狗的血清是否与所述蛋白反应。
20.如权利要求17所述的方法，该方法还包括测定所述狗的血清是否与犬埃利希氏体的p28蛋白反应的步骤，其中与p153蛋白和p28两种蛋白发生免疫反应表示所述狗感染了犬埃利希氏体。
21.一种测定狗是否感染了埃利希氏体的血清学诊断试剂盒，其特征在于，所述试剂盒包含：
a)一种或多种选自p153、p43、p156或p28的固定化的埃利希氏体的抗原；
b)狗血清的合适稀释缓冲液；
c)连接有报告分子的抗狗血清第二抗体；和，
d)检测所述报告分子的合适的试剂。
22.如权利要求21所述的试剂盒，其中所述的埃利希氏体的抗原被固定在膜或微量滴定板上。
23.如权利要求21所述的试剂盒，其中，所述的报告分子选自荧光素酶、辣根过氧化酶、β-半乳糖苷酶和荧光标记物。
24.一种测定狗是否感染了埃利希氏体的方法，其特征在于，该方法包括以下步骤：
提取所述狗血的DNA；和
用犬埃利希氏体p153基因(或恰菲埃利希氏体p156基因)的特异性寡核苷酸引物对所述DNA进行PCR扩增；
根据大小分离所得的PCR产物，其中对适当大小的扩增产物得到阳性检测结果就表示检测对象感染了犬埃利希氏体或恰菲埃利希氏体。
25.如权利要求24所述的方法，其中所述的PCR产物采用凝胶电泳进行检测。
26.一种测定狗是否感染了埃利希氏体的试剂盒，其特征在于，所述试剂盒包含：
a)提取血液中DNA所用的试剂；
b)p153-特异性或p156-特异性寡核苷酸；和
c)PCR扩增所用的试剂。
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| CN102834411A (zh) * | 2009-11-20 | 2012-12-19 | 爱贝斯股份有限公司 | 用于检测埃里希体属抗体的肽、装置和方法 |
| CN103200960A (zh) * | 2010-07-02 | 2013-07-10 | 英特维特国际股份有限公司 | 针对犬埃利希体的疫苗和相关方法 |
| US9157913B2 (en) | 2012-10-11 | 2015-10-13 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
| US9442112B2 (en) | 2014-04-04 | 2016-09-13 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
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| US7842473B2 (en) * | 2005-04-04 | 2010-11-30 | Idexx Laboratories, Inc. | Ehrlichia canis DIVA (differentiate infected from vaccinated animals) |
| US7842474B2 (en) | 2005-04-04 | 2010-11-30 | Idexx Laboratories, Inc. | Ehrlichia canis DIVA (differentiate infected from vaccinated animals) |
| BRPI0611820B8 (pt) | 2005-06-16 | 2021-05-25 | Res Found Dev | método de identificar uma infecção por e. canis ou e. chaffeensis |
| PT2187958T (pt) | 2007-09-11 | 2017-03-17 | Yissum Res Dev Company Of The Hebrew Univ Of Jerusalem | Vacina atenuada de erliquiose |
| WO2009039414A2 (en) | 2007-09-21 | 2009-03-26 | Idexx Laboratories, Inc. | Methods and compositions for detection of ehrlichia chaffeensis (vlpt) |
| EP2045816A1 (en) * | 2007-10-01 | 2009-04-08 | Paul Scherrer Institut | Fast readout method and swiched capacitor array circuitry for waveform digitizing |
| EP3527221B1 (en) | 2007-10-31 | 2022-02-23 | Idexx Laboratories, Inc. | Ehrlichia canis diva (differentiate infected from vaccinated animals) |
| KR101588736B1 (ko) | 2008-01-10 | 2016-01-26 | 리서치 디벨롭먼트 파운데이션 | 얼리키아 샤피엔시스에 대한 백신 및 진단제 |
| US9194870B2 (en) | 2014-01-21 | 2015-11-24 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Anaplasma antibodies |
| EP3996741A1 (en) * | 2019-07-12 | 2022-05-18 | Research Development Foundation | Ehrlichia vaccines and immunogenic compositions |
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| US6653128B2 (en) * | 1996-10-17 | 2003-11-25 | University Of Florida | Nucleic acid vaccines against rickettsial diseases and methods of use |
| US6207169B1 (en) * | 1997-03-21 | 2001-03-27 | Corixa Corporation | Compounds and methods for the diagnosis and treatment of Ehrlichia infection |
| US6403093B1 (en) * | 1997-03-21 | 2002-06-11 | Mayo Foundation For Medical Education And Research | Methods to detect granulocytic ehrlichiosis |
| WO1999052370A1 (en) * | 1998-04-09 | 1999-10-21 | The Ohio State Research Foundation | Nucleic acids encoding outer membrane protein of human granulocytic ehrlichiosis agent |
| US6436399B1 (en) * | 1998-04-09 | 2002-08-20 | The Ohio State University Research Foundation | Nucleic acid encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby |
| US6043085A (en) * | 1998-08-27 | 2000-03-28 | Research Development Foundation | Ehrlichia canis 120-kDa immunodominant antigenic protein and gene |
| US6544517B1 (en) * | 1998-09-18 | 2003-04-08 | The Ohio State University Research Foundation | Outer membrane protein of Ehrlichia canis and Ehrlichia chaffeensis |
| US6355777B1 (en) * | 2000-04-28 | 2002-03-12 | Research Development Foundation | P43 antigen for the immunodiagnosis of canine ehrlichiosis and uses thereof |
| US6512005B2 (en) | 2001-02-28 | 2003-01-28 | Taro Pharmaceutical Industries, Ltd. | Process for synthesis of pure warfarin acid, warfarin alkali metal salts and corresponding clathrates |
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| US9470682B2 (en) | 2009-11-20 | 2016-10-18 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
| US8828675B2 (en) | 2009-11-20 | 2014-09-09 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
| CN102834411A (zh) * | 2009-11-20 | 2012-12-19 | 爱贝斯股份有限公司 | 用于检测埃里希体属抗体的肽、装置和方法 |
| CN102834411B (zh) * | 2009-11-20 | 2015-10-21 | 爱贝斯股份有限公司 | 用于检测埃里希体属抗体的肽、装置和方法 |
| CN103200960A (zh) * | 2010-07-02 | 2013-07-10 | 英特维特国际股份有限公司 | 针对犬埃利希体的疫苗和相关方法 |
| CN103200960B (zh) * | 2010-07-02 | 2019-04-23 | 英特维特国际股份有限公司 | 针对犬埃利希体的疫苗和相关方法 |
| US9157913B2 (en) | 2012-10-11 | 2015-10-13 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
| US9651546B2 (en) | 2012-10-11 | 2017-05-16 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
| US9696300B2 (en) | 2012-10-11 | 2017-07-04 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
| US10444231B2 (en) | 2012-10-11 | 2019-10-15 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
| US10948487B2 (en) | 2012-10-11 | 2021-03-16 | Zoetis Services Llc | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
| US9851352B2 (en) | 2014-04-04 | 2017-12-26 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
| US9442112B2 (en) | 2014-04-04 | 2016-09-13 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
| US11204351B2 (en) | 2014-04-04 | 2021-12-21 | Zoetis Services Llc | Compositions and methods for identifying Ehrlichia species |
Also Published As
| Publication number | Publication date |
|---|---|
| DE60316189T2 (de) | 2007-12-20 |
| AU2003290575A1 (en) | 2004-06-07 |
| CA2504762A1 (en) | 2004-05-21 |
| CN101294162B (zh) | 2011-03-30 |
| EP1581638B1 (en) | 2007-09-05 |
| US7204992B2 (en) | 2007-04-17 |
| EP1581638A4 (en) | 2005-12-28 |
| ES2293057T3 (es) | 2008-03-16 |
| JP4494974B2 (ja) | 2010-06-30 |
| KR101063177B1 (ko) | 2011-09-08 |
| JP2006505270A (ja) | 2006-02-16 |
| US20070218082A1 (en) | 2007-09-20 |
| WO2004042037A1 (en) | 2004-05-21 |
| KR20050084679A (ko) | 2005-08-26 |
| US20040121433A1 (en) | 2004-06-24 |
| CN101294162A (zh) | 2008-10-29 |
| US7754224B2 (en) | 2010-07-13 |
| DE60316189D1 (de) | 2007-10-18 |
| BRPI0315980B1 (pt) | 2016-03-29 |
| MXPA05004804A (es) | 2005-12-05 |
| CN100386430C (zh) | 2008-05-07 |
| AU2003290575B2 (en) | 2009-10-29 |
| EP1581638A1 (en) | 2005-10-05 |
| ATE372378T1 (de) | 2007-09-15 |
| BR0315980A (pt) | 2005-09-20 |
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